Priapism associated with sickle cell disease affects several thousand men in the US and millions worldwide. At present there is no pharmacological treatment to prevent the onset of priapism in these patients, in part because the mechanisms involved in the development of priapism are complex and not well understood. Our group has established that a family of pentapeptides called opiorphins play a role in the development of priapism. Our most recent work has discovered that opiorphins are up-regulated in corporal smooth tissue in response to hypoxia and that opiorphins then regulate expression of genes that function in smooth muscle relaxant pathways. This has led us to propose the following hypothesis: Sickle cell disease causes hypoxia/ischemia in corporal tissue which results in increased opiorphin expression. Opiorphin then acts as a master regulator of compensatory smooth muscle relaxant pathways. The excessive activation of these relaxant pathways in corporal tissue is generally considered to lead to priapism. Our hypothesis will be tested in three Specific Aims. In Specific Aim 1 we will determine if opiorphin expression is increased in response to hypoxia in mouse and human cells in vitro and if up-regulation of opiorphin expression in turn activates pathways involved in corporal smooth muscle relaxation. We will confirm that the in vitro response to hypoxia correlates to similar changes in relaxant pathways in an in vivo corporal mouse model of hypoxia and if in this animal model there is heightened relaxation of corporal smooth muscle strips in organ bath studies. In Specific Aim 2 we will focus on a mouse model of sickle cell disease and determine if up-regulation of opiorphin expression in corporal tissue is an early event in sickle cell disease. We will determine in the sickle cell mouse corporal tissue if a similar set of genes involved in relaxant pathways are activated as in models of hypoxia (identified in Specific Aim 1). We will correlate changes in the genes involved in relaxant pathways with increased erectile tendencies and heighted relaxation of corporal smooth muscle tissue. In addition, we will determine changes in gene expression in a chemically-(blebbistatin)-induced mouse model of priapism. Analysis of the blebbistatin mouse model will distinguish genes that are changed secondary (ie. in response to priapism) rather than causative of priapism. In a clinical study we will compare the expression of opiorphin in the blood of sickle cell and non-sickle cell patients, and determine if opiorphin levels are increased during, or predictive of, a priapic-crisis. In Specific Aim 3 will develop opiorphin over expression or knock-down mouse models to directly demonstrate that opiorphins play a role in priapism/erectile function. These studies will utilize a novel delivery system developed by Dr. Joel Friedmans (Co-I) at Einstein- a paramagnetic nanoparticle carrying opiorphin-siRNA which can be targeted to the penis. We will use this system to specifically knock-down expression of opiorphin in the corporal tissue of sickle cell mice and determine if knock-down of opiorphin expression results in decreased erectile tendencies or decreased activity of smooth muscle relaxant pathways. If evidence confirms that knock-down of opiorphin reduces priapic-like activity in this animal model it would provide proof-of-principle to support this approach, or other strategies targeting opiorphin, to prevent priapism in sickle cell patients.